The high affinity of Paracoccus denitrificans cells for nitrate as an electron acceptor. Analysis of possible mechanisms of nitrate and nitrite movement across the plasma membrane and the basis for inhibition by added nitrite of oxidase activity in permeabilised cells

Abstract

(1) The apparent K m for nitrate of the electron-transport system in intact cells of Paracoccus denitrificans was less than 5 μM. In contrast the apparent K m for nitrate of inverted membrane vesicles oxidising NADH was greater than 50 μM. When azide, a competitive inhibitor, was present, the apparent K m observed with the vesicles was raised to 0.64 mM, consistent with values previously reported for purified preparations of the reductase. In membrane vesicles the nitrate reductase is probably not rate-limiting for NADH-nitrate oxido-reductase activity, and thus a lower limit for K m (NO 3 −) is obtained. It is suggested that the very low K m (NO 3 −) in intact cells must arise from either a transport process or a nitrate-specific pore that allows access of nitrate directly to the active site of its reductase from the periplasm. (2) The swelling of spheroplasts has been studied under both aerobic and anaerobic conditions to probe possible mechanisms of nitrate and nitrite transport across the plasma membrane of P. denitrificans. Nitrate reductase was inhibited by azide to prevent reduction of internal nitrate. No evidence for operation of either nitrate-nitrite antiport or proton-nitrate symport was obtained. (3) Measurements from the fluorescence intensity of 8-anilino-naphthalene-1-sulphonate of the rates of decay of diffusion potentials generated by addition of potassium salts to valinomycin-treated plasma membrane vesicles from P. denitrificans showed that the permeability of the membrane to anions is SCN − > NO 3 −, NO 2 −, pyruvate, acetate > CI − > SO 4 2−. In the presence of a protonophore the rate of decay of the diffusion potential was considerably enhanced with potassium acetate or potassium nitrite, but not with potassium salts of nitrate, chloride or pyruvate. This result indicates that HNO 2 and CH 3COOH can rapidly and passively diffuse across the cell membrane. This finding suggests that transport systems for nitrite are in general probably not required in bacteria. The failure of a protonophore to enhance the dissipation of the diffusion potential generated by potassium nitrate is evidence against the operation of a proton-nitrate symporter. (4) Low concentrations of added nitrite very strongly inhibit electron flow to oxygen in anaerobically grown cells, provided that they have been treated with Triton X-100 or an uncoupler. This inhibition is not observed with aerobically grown cells. It is concluded that the inhibitory species is a reaction product or an intermediate of the nitrite reductase reaction. The requirement for collapse of protonomotive force by uncoupler or permeabilising the plasma membrane suggests that any such species could be negatively charged. Nitroxyl anion (NO −) can be considered, as its conjugate acid is a postulated intermediate between nitrite and nitrous oxide; nitroxyl anion can bind to heme centres to give nitrosyl derivatives. (5) The basis for the ability of permeabilised, but not intact, cells of P. denitrificans to reduce oxygen and nitrate simultaneously is discussed.